System for achieving high expression of genes

ABSTRACT

The present invention relates to a method of expressing a gene by inserting the genome into a host organism using genetic engineering techniques. The present invention further relates to a novel promoter, a recombinant vector containing the promoter and a target gene, a transformant containing the recombinant vector, and a method of producing a useful gene product or useful substance using the transformant.

FIELD OF THE INVENTION

The present invention relates to a method of expressing a gene byinserting the genome into a host organism using a genetic engineeringtechnique. The present invention further relates to a novel promoter, arecombinant vector containing the promoter and a target gene, atransformant containing the recombinant vector, and a method ofproducing a useful gene product or a useful substance using thetransformant.

BACKGROUND ART

When substances are produced by a host organism, or the function of ahost organism is altered or analyzed, a genetic engineering technique isemployed. This involves introducing a homologous or heterologous geneinto the host organism for expression. However, this genetic engineeringtechnique is not sufficient in terms of stability and high expression,and thus there have been expectations that it would be improved.

For example, when a host organism is yeast Saccharomyces cerevisiae, aYEP type vector utilizing a 2-μm DNA is often used. The YEP vectorenables introduction of a large number of copies of a gene. However, theYEP vector cannot be said to be sufficient in terms of stability becausean enzyme activity of the product from the introduced gene may decreasedue to, for example, the loss of the vector during cell division. Toimprove the enzyme activity, a vector is designed to contain a drugresistance marker and the drug is added to a medium, or designed tocontain an auxotrophic marker when the auxotrophic marker has alreadybeen provided in a host strain, and selection pressure can be applied byfurther utilizing a highly purified minimum medium (YNB, Difco) as amedium. However, all such cases are disadvantageous in that the mediumcost is expensive.

In the meantime, as a vector that can integrate a gene into achromosome, a YIP type vector utilizing homologous recombination isknown. This YIP vector enables a transgene to be present stably on thegenome depending on the design of the vector, but in general it isunable to achieve high expression of the transgene.

From the reasons described above, a method of highly expressing a geneby introducing or inserting the gene into a host organism stably and atlow cost has been desired in the art.

Furthermore, for example when a target gene is expressed in a yeast cellSaccharomyces cerevisae, it is required to ligate a promoter that can beexpressed within yeast upstream of the gene. As a currently reportedpromoter for Saccharomyces cerevisae, the promoter of the alcoholdehydrogenase 1 (ADH1) gene and the promoter of the 3-phosphoglyceratekinase (PGK) gene are known to show strong expression levels. Moreover,gene transfer by homologous recombination leads to high stability of thegene. Hence, if a target gene can be expressed under a strong promoter,it would be efficient in producing a substance, and altering andanalyzing the function.

However, when a gene is inserted into yeast by homologous recombination,the above YIP vector is utilized. In this case, the number of copiesthat can be expected is only 1 or 2. Thus, development of a promoterthat enables high expression even with a single copy has been desired.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of highlyexpressing a target gene by stably introducing the target gene into ahost organism. Furthermore, another object of the present invention isto provide a promoter having strong transcription activity for theexpression of a target gene, and a recombinant vector containing thepromoter, a transformant containing the recombinant vector, and a methodof producing the expression product of a target gene or a usefulsubstance using the transformant.

As a result of thorough studies to achieve the above objects, we havefound that a target gene can be stably and highly expressed by ligatingthe target gene to be expressed to a pyruvate decarboxylase 1 promoterin yeast Saccharomyces cerevisae, and then integrating the gene into thegenome of a host.

Furthermore, we have focused on the event that an extremely largequantity of ethanol is produced in Saccharomyces cerevisae. We haveexpected that the pyruvate decarboxylase 1 gene is highly expressed inthe ethanol fermentation pathway, and isolated the promoter region ofthe above pyruvate decarboxylase 1 (PDC1) gene. Furthermore, we haveligated the promoter region of PDC1 gene to a target gene, and insertedthe gene into Saccharomyces cerevisae, thereby obtaining a finding thatthe target gene is highly expressed under the promoter. Based on theabove findings, we have completed the present invention.

Specifically, the present invention relates to a method of expressing agene, which comprises inserting a target gene into a genome under thecontrol of the promoter of a gene wherein an autoregulation mechanism ispresent, or the promoter of a gene that is not essential for growth orfermentation in a host organism. The promoter may be a DNA that containsa sequence derived from the nucleotide sequence of the promoter of agene wherein an autoregulation mechanism is present, or the nucleotidesequence of the promoter of a gene that is not essential for growth orfermentation in a host organism by deletion, substitution or addition of1 to 40 nucleotides, and has promoter activity. Moreover the promotermay be a DNA that is capable of hybridizing under stringent conditionsto a DNA comprising a sequence complementary to the whole or a part ofthe nucleotide sequence of the promoter of a gene wherein anautoregulation mechanism is present, or the nucleotide sequence of thepromoter of a gene that is not essential for growth or fermentation in ahost organism, and has promoter activity.

In the present invention, the above promoter of a gene wherein theautoregulation mechanism is present includes, for example, the promoterof the pyruvate decarboxylase 1 gene. The promoter of a gene that is notessential for growth includes, for example, the promoter of a geneencoding thioredoxin.

In this case, a host organism may be any of bacteria, yeast, insects,animals or plants. In particular, yeast belonging to the genusSaccharomyces is preferred. These host organisms also mean any ofindividual organisms (excluding humans), tissues and cells.

Furthermore, in the present invention, the above promoter of thepyruvate decarboxylase 1 gene is a promoter comprising any one of thefollowing DNAs (a) to (c):

(a) a DNA, comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA, comprising a nucleotide sequence derived from the nucleotidesequence represented by SEQ ID NO: 1 by deletion, substitution oraddition of 1 to 40 nucleotides, and having promoter activity; and

(c) a DNA, capable of hybridizing under stringent conditions to a DNAcomprising a sequence complementary to the whole or a part of thenucleotide sequence represented by SEQ ID NO: 1, and having promoteractivity.

The present invention also relates to a recombinant vector containingthe above promoter. It is preferred that the recombinant vector of thepresent invention is operably linked a target gene. In this case, therecombinant vector may be a plasmid vector or a viral vector. Inaddition, a target gene in the recombinant vector is, for example, anucleic acid selected from a nucleic acid encoding a protein or theantisense nucleic acid thereof, a nucleic acid encoding an antisense RNAdecoy and a ribozyme.

Furthermore, the present invention provides a transformant that isobtainable by transforming a host using any one of the above recombinantvectors. Hosts used herein can be bacteria, yeast, animals, insects orplants. In particular, a host is preferably yeast belonging to the genusSaccharomyces. These host organisms also mean any of individualorganisms (excluding humans), tissues and cells.

Furthermore, the present invention provides a method of producing theexpression product of a target gene or a substance produced by theexpression product, which comprises culturing any one of the abovetransformants in a medium, and collecting the expression product of atarget gene or a substance produced by the expression product from theobtained culture product.

The present invention is explained in detail as follows. Thisapplication claims a priority from Japanese Patent Application No.2001-286637 filed Sep. 20, 2001 and Japanese Patent Application No.2002-128323 filed Apr. 30, 2002 which claims a priority from theaforementioned application, and Japanese Patent Application No.2001-287159 filed Sep. 20, 2001, and Japanese Patent Application No.2002-128286 filed Apr. 30, 2002, which claims a priority from theaforementioned application. This application includes the content asdisclosed in the specifications and/or drawings of the above JapanesePatent Applications.

In the living world, some have genes wherein an autoregulation mechanismis present and genes that are not essential for growth or fermentation.We have focused on this point, and selected promoters of such genes formethods of gene transfer and gene expression. Hence, the gene expressionmethod of the present invention comprises inserting a target gene into agenome under the control of the promoter of a gene wherein anautoregulation mechanism is present or the promoter of a gene that isnot essential for growth or fermentation in a host organism. The methodof the present invention is as summarized as follows.

1. Selection of Promoter

First, the promoter of a gene wherein an autoregulation mechanism ispresent, or the promoter of a gene that is not essential for growth orfermentation of a host organism is selected. As a target host organism,all organisms that are expected to produce substances, and to altertheir function or to analyze their function, can be used as hostorganisms. Examples of a host organism include bacteria, yeast, insects,animals and plants.

(1) Gene Promoter wherein an Autoregulation Mechanism is Present

To select the promoter of a gene wherein an autoregulation mechanism ispresent, a gene wherein the autoregulation mechanism is present is firstspecified. “Autoregulation mechanism” means a mechanism wherein aplurality of genes having the same function is present in the sameorganism, of which at least one gene is normally expressed and theremainders are suppressed, and the remaining genes are expressed tocontinue the function only when the generally expressed gene becomesunable to function because of disruption or the like. For example, thepyruvate decarboxylase (PDC) gene of yeast belonging to the genusSaccharomyces is a gene encoding an enzyme that converts bydecarboxylating pyruvic acid into acetaldehyde in the process of ethanolsynthesis, and plays an important role in the fermentation process. PDCincludes PDC1, PDC5 and PDC6, but normally PDC1 is activated, and PDC5and PDC6 are suppressed by the action of PDC1. However, when genedisruption or a mutation caused by a drug occurs to PDC1 so as toinactivate the function thereof, the PDC5 gene is activated, whereby theethanol-producing function of yeast is not lost (Eberhardt, I. et al.,Eur. J. Biochem. 1999, 262(1): 191-201; Muller, E H. et al., FEBS Lett.1999, 449 (2-3): 245-250). Actually, Schaaff et al., have deleted thePDC1 promoter, and then confirmed the pyruvate decarboxylase activity ofyeast (Schaaff, I. et al., Curr. Genet. 1989, 15:75-81). Specifically,the phenotype is almost equivalent to that of the parent strain. On theother hand, the PDC 1 promoter has been isolated in the genusKluyveromyces classified as yeast (International publication WO94/01569). However, the autoregulation mechanism has not been reportedin the genus Kluyveromyces.

A gene wherein the autoregulation mechanism is present can be specifiedby confirming, when a gene is disrupted in a strain, if a proteinencoded by the gene is still expressed. The promoter of the thusspecified gene wherein the autoregulation mechanism is present isselected. In the present invention, for example, the promoter of PDC1(hereinafter referred to as the PDC1 promoter) can be selected.

(2) Promoter of Gene that is not Essential for Growth or Fermentation

To select the promoter of a gene that is not essential for growth orfermentation of a host organism, a gene that is not essential for growthor fermentation is first specified. Here, “growth” means that cellssurvive such that they can proliferate. “Fermentation” means alcoholfermentation or the like whereby substances are produced. Furthermore,“gene that is not essential” means a gene that is not involved in theprocesses of growth or the fermentation, such that even when the gene isdisrupted or inactivated, the growth, the fermentation or both are stillmaintained.

A gene that is not essential for growth or fermentation, can bespecified by confirming if a host organism of the strain, wherein a geneis disrupted, continues its growth and fermentation. An example of sucha gene is the TRX1 gene encoding thioredoxin, which is present in mostorganisms. The TRX1 gene is involved in DNA replication, oxidativestress response, the heredity of vacuole and the like, but is not alwaysessential for growth or fermentation. Specifically, if the TRX1 gene isdisrupted or substituted with other genes in a host organism, the hostorganism can continue the growth or the fermentation. The promoter ofthe thus specified gene, which is not essential for growth orfermentation of the host organism, is selected.

2. Preparation of Target Gene and Promoter

The above-selected promoter and a target gene to be inserted into a hostorganism are prepared. In the present invention, “target gene” means agene, the expression of which is desired in order to produce a substanceand to alter or analyze the function, and may be either a homologous orheterologous gene. For example, for the purpose of substance production,a gene encoding a useful protein is preferred as a target gene. Examplesof such a useful protein include interferons, vaccines and hormones.Moreover, a target gene may also be a gene encoding an enzyme producinga useful substance. An example of such a gene is a gene encoding lactatedehydrogenase, which produces lactic acid from pyruvic acid.

For the preparation of a target gene and the above promoter, anytechnique known in the art can be employed. For example, when a targetgene and the above promoter are isolated from a source, a target geneand the above promoter can be prepared by a method for synthesizing cDNAfrom RNA that has been prepared by a guanidine isothiocyanate method. Inaddition, a target gene and the above promoter can also be prepared byamplification by PCR using a genomic DNA as a template. The thusobtained DNAs of a target gene and the above promoter can be useddirectly depending on the purpose, or can be used after digestion with arestriction enzyme or after addition of a linker, if desired.

In the present invention, a DNA containing a sequence derived from thenucleotide sequence of the promoter by deletion, substitution oraddition of 1 to 40 nucleotides, and having promoter activity, can alsobe utilized as a promoter. Promoter activity means to have the abilityand function of causing a target gene to produce gene products in a hostor outside the host when the target gene is inserted into the host byoperably linking the target gene downstream to the promoter. In such aDNA, the promoter activity is maintained at a level that enables almostthe same application thereof under the same conditions as those for apromoter comprising a full-length nucleotide sequence without anymutations (deletion, substitution or addition) to function. For example,such a DNA maintains promoter activity that is approximately 0.01 to 100times, preferably approximately 0.5 to 20 times, and more preferablyapproximately 0.5 to 2 times greater than that of a full-lengthsequence.

Such a DNA can be produced as described in literature such as MolecularCloning (Sambrook, et al., ed., (1989) Cold Spring Harbor Lab. Press,New York).

For example, by the technology in 1 to 40 nucleotides is(are) deleted,substituted or added based on and from the nucleotide sequence of thepromoter of a gene wherein the autoregulation mechanism is present, orthe nucleotide sequence of the promoter of a gene that is not essentialfor growth or fermentation in a host organism, for example by thesite-directed mutagenesis method, a variant having a different sequencewhile maintaining promoter activity can be prepared. For example, forsite-directed mutagenesis whereby 1 to 40 nucleotides are substituted, avariant can be obtained according to the technology described inliterature such as Proc. Natl. Acad. Sci. USA 81 (1984) 5662-5666;International Publication No. WO85/00817; Nature 316 (1985) 601-605;Gene 34 (1985) 315-323; Nucleic Acids Res. 13 (1985) 4431-4442; Proc.Natl. Acad. Sci. USA 79(1982) 6409-6413; or Science 224 (1984)1431-1433, and then the variant can be utilized. In addition, thesevariants can be prepared using a commercially available kit (Mutan-G andMutan-K (TAKARA BIO)). Furthermore, error-prone polymerase chainreaction (error-prone PCR) is also known as a method for preparingvariants, and by selecting a condition wherein the degree of accuracyfor replication is low, a mutation of 1 to several nucleotides can beintroduced (Cadwell, R. C. and Joyce, G. F. PCR Methods and Applications2(1992) 28-33; Malboeuf, C. M. et al. Biotechniques 30(2001) 1074-8;Moore, G. L. and Maranas C. D. J. Theor. Biol. 7; 205 (2000) 483-503).

Furthermore, hybridization under stringent conditions using, as a probe(100 to 900 nucleotides), a DNA comprising a sequence complementary tothe whole or a part of the nucleotide sequence of the promoter of a genewherein the autoregulation mechanism is present, or the promoter of agene that is not essential for growth or fermentation in a host organismenables to newly obtain and utilize a DNA that has a function (that is,promoter activity) similar to that of a DNA comprising the nucleotidesequence of the promoter of a gene wherein the autoregulation mechanismis present, or the promoter of a gene that is not essential for growthor fermentation in a host organism, and comprises another nucleotidesequence. Here, stringent conditions mean conditions wherein, forexample, the sodium concentration is between 10 and 300 mM, andpreferably between 20 and 100 mM, and the temperature is between 25 and70° C., and preferably between 42 and 55° C.

Whether or not a variant obtained as described above and a DNA obtainedby hybridization have activity as promoters can be confirmed by thefollowing procedures. Specifically, the promoter activity of the DNAobtained as described above can be confirmed by preparing a vectorwherein, preferably, various reporter genes, for example, the luciferase(LUC) gene, the chloramphenicol acetyltransferase (CAT) gene and theβ-galactosidase (GAL) gene are ligated to the downstream region of thepromoter, inserting the genes into a host genome using the vector, andthen measuring the expression of the reporter genes.

3. Insertion of Target Gene and Promoter

Subsequently, the above gene wherein the autoregulation mechanism ispresent, or a gene that is not essential for growth or fermentation in ahost organism is disrupted, and then a target gene is inserted under thecontrol of the promoter of the gene, or alternatively, the gene issubstituted with a target gene.

For example, the target gene isolated as described above is operablylinked to the promoter selected as described above, and then insertedinto the genome of a host organism. “Operably linked to” means that atarget gene is linked to the above promoter so that the target gene isexpressed under the control of the above promoter in a host organism towhich the target gene is inserted. A target gene and the above promotercan be inserted using any technique known in the art. For example, atarget gene and the above promoter can be inserted into the genome of ahost organism using a recombinant vector. A recombinant vector can beobtained by ligating (inserting) a target gene and the above promoter toan appropriate vector. Examples of a vector for the insertion of atarget gene are not specifically limited, as long as they can beintegrated into the genome in a host organism, and include a plasmidDNA, a bacteriophage DNA, a retrotransposon DNA and a yeast artificialchromosome DNA (YAC).

Examples of a plasmid DNA include YIp-type Escherichia coli-yeastshuttle vectors such as pRS403, pRS404, pRS405, pRS406, pAUR101 orpAUR135; plasmids derived from Escherichia coli (ColE plasmid such aspBR322, pBR325, pUC18, pUC19, pUC118, pUC119, pTV118N, pTV119N,pBluescript, pHSG298, pHSG396 or pTrc99A; a p15A plasmid such aspACYC177 or pACYC184; or a pSC101 plasmid such as pMW118, pMW119, pMW218or pMW219); and plasmids derived from Bacillus (e.g., pUB110 or pTP5).Examples of a phage DNA include λ phage (Charon4A, Charon21A, EMBL3,EMBL4, ?gt10, ?gt11 or ?ZAP), fX174, M13mp18 and M13mp19. An example ofretrotransposon is Ty factor. An example of a vector for YAC is pYACC2.

To insert a target gene and the above promoter into a vector, forexample, a method that is employed herein involves, first, cleaving apurified DNA with an appropriate restriction enzyme, and then insertingthe product at the restriction site or the multi-cloning site of anappropriate vector DNA so as to ligate the product to the vector.

A target gene should be incorporated into a vector so that the functionof the gene is exerted under the control of the above-selected promoter.Hence, in addition to the above-selected promoter, a target gene and aterminator, cis element such as an enhancer, splicing signal, polyAaddition signal, ribosome binding sequence (SD sequence) and the likecan be ligated to a recombinant vector, if desired. Furthermore, aselection marker indicating that the vector is retained within the cellmay also be ligated. In addition, examples of a selection marker includethe dihydrofolate reductase gene, the ampicillin resistance gene and theneomycin resistance gene. In addition, an example of a marker gene isthe gene for tryptophan synthesis (TRP1 gene), but is not limitedthereto. Other marker genes, for example, the URA3 gene, the ADE2 geneand the HIS3 gene having auxotrophic ability, or the G418 resistancegene having drug resistance ability can also be utilized.

An example of a terminator sequence is the terminator gene of theglyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), but is notlimited thereto in the present invention. Any terminator sequence may beused, as long as it is a terminator sequence that can be used within ahost organism.

As described above, a recombinant vector can be prepared so as to beapplicable for the expression of a target gene in a host organism. Bytransforming a host organism using the recombinant vector, a target genecan be expressed under the control of the above-selected promoter in thehost organism.

When a bacterium such as Escherichia coli is used as a host, arecombinant vector is preferably composed of a promoter, a ribosomebinding sequence, a target gene and a transcription terminationsequence. In addition, a gene regulating a promoter may also becontained.

Examples of Escherichia coli include Escherichia coli K12 and DH1. Anexample of Bacillus is Bacillus subtilis. A method for introducing arecombinant vector into bacteria is not specifically limited, as long asit is a method for introducing a DNA into bacteria. Examples of such amethod include a method using calcium ions and an electroporationmethod.

When yeast is used as a host, for example, Saccharomyces cerevisiae,Schizosaccharomyces pombe or Pichia pastoris can be used. A method forintroducing a recombinant vector into yeast is not specifically limited,as long as it is a method for introducing a DNA into yeast. Examples ofsuch a method include an electroporation method, a spheroplast methodand a lithium acetate method.

When an insect or an animal is used as a host, for example, a calciumphosphate method, a lipofection method or an electroporation method maybe employed as a method for introducing a recombinant vector into thehost.

When a plant is used as a host, for example, an agrobacterium method, aparticle gun method, a PEG method or an electroporation method may beemployed as a method for introducing a recombinant vector into the host.

When an insect, an animal (excluding a human) or a plant individual isused as a host, a recombinant vector can be introduced according to atechnique known in the art for generating a transgenic animal or plant.Examples of a method for introducing a recombinant vector into an animalindividual include a method for microinjection into fertilized eggs, amethod for introduction into ES cells, and a method for introducing acell nucleus that has been introduced into a culture cell into afertilized egg by nuclear transplantation.

A host organism wherein a recombinant vector is introduced as describedabove is subjected to selection for strains (clones) having a targetgene introduced under the control of the above-selected promoter.Specifically, transformant are selected using the above selection markeras an indicator.

Whether or not a target gene is incorporated under the control of theabove promoter can be confirmed by the PCR (polymerase chain reaction)or the Southern hybridization. For example, a DNA is prepared from atransformant, introduced DNA-specific primers are designed, and then PCRis performed using the primers and prepared DNA. Subsequently, theamplification product is subjected to agarose gel electrophoresis,polyacrylamide gel electrophoresis or capillary electrophoresis, stainedwith ethidium bromide, SYBR Green solution or the like and then detectedas a single band, so that the introduced DNA can be confirmed.Furthermore, PCR is performed using primers previously labeled withfluorescent dye or the like, so that an amplification product can bedetected. Furthermore, a method that can be also employed hereininvolves binding an amplification product to a solid phase such as amicroplate, and then confirming the amplification product byfluorescence reaction, enzyme reaction or the like.

As described above, under the control of the promoter of a gene whereinthe autoregulation mechanism is present, or the promoter of a gene thatis not essential for growth or fermentation of a host organism, a targetgene is inserted into a genome (genome integration), so that the targetgene is expressed in the host organism. Since the PDC1 promoter is avery strong promoter, when the PDC1 promoter is selected, a target geneis highly expressed even when inserted in the form of a single copy intothe genome. Furthermore, since the endogenous gene of which the promoteris selected is not essential for growth and fermentation, even when itis disrupted or substituted with a target gene, the host organism cancontinue the growth and the fermentation so as to be able to express thetarget gene for a long time period.

4. Pyruvate Decarboxylase 1 Gene (PDC1) Promoter

The promoter of the present invention is a promoter (hereinafter,referred to as the PDC1 promoter) of pyruvate decarboxylase 1 geneisolated from Saccharomyces cerevisiae. Pyruvate decarboxylase is anenzyme involved in the ethanol fermentation pathway of yeast. Ingeneral, only PDC1 functions among PDC1, PDC5 and PDC6 genes (see “1.Selection of promoter” section). We focused on the fact that althoughpyruvate decarboxylase is produced by the expression of only PDC1because of the autoregulation mechanism, ethanol is produced in a largequantity, and then specified the promoter region of PDC1.

The PDC1 promoter was determined and isolated as described below. Firstby the use of the public genome database of Saccharomyces cerevisiae(Saccharomyces Genome Database), a vector for homologous recombinationwas constructed so that a target gene could be inserted downstream ofPDC1 promoter. This vector was introduced, strains with high expressionamounts of the target gene were selected, PDC1 promoter fragments wereobtained by PCR, and then the nucleotide sequence of a putative regioncorresponding to PDC1 promoter was determined by a sequencer (ABI 310Genetic Analyzer).

The PDC1 promoter contains a DNA comprising the nucleotide sequencerepresented by SEQ ID NO: 1. After isolation of the PDC1 promoter, theDNA can be obtained by chemical synthesis according to a technique fornucleic acid synthesis.

Moreover, the PDC1 promoter of the present invention also includes a DNAcomprising a nucleotide sequence isolated from the nucleotide sequencerepresented by SEQ ID NO: 1 by deletion, substitution or addition of 1to 40 nucleotides, and having promoter activity. Promoter activity meansto have the ability and function of producing the gene product of atarget gene within a host or outside a host when the target gene isinserted into a host by operably linked the target gene downstream tothe promoter. In such a DNA, the promoter activity is maintained at alevel that enables almost the same applications thereof under the sameconditions as those for a promoter comprising the nucleotide sequencerepresented by SEQ ID NO: 1 to function. For example, such a DNAmaintains promoter activity that is approximately 0.01 to 100 times,preferably approximately 0.5 to 20 times, and more preferablyapproximately 0.5 to 2 times greater than that of the DNA comprising thenucleotide sequence represented by SEQ ID NO: 1.

Such a DNA can be produced as described in literature such as MolecularCloning (Sambrook et al., ed., (1989) Cold Spring Harbor Lab. Press, NewYork) by referring to the nucleotide sequence represented by SEQ ID NO:1.

For example, by the technology in which 1 to 40 nucleotides is(are)deleted, substituted or added based on and from the above-describednucleotide sequence represented by SEQ ID NO: 1, such as thesite-directed mutagenesis method as described in the above “2.Preparation of target gene and promoter” section, a variant having adifferent sequence can be prepared while maintaining promoter activity.

Furthermore, hybridization under stringent conditions using, as a probe(100 to 900 nucleotides), a DNA comprising a sequence complementary tothe whole or a part of the nucleotide sequence represented by SEQ ID NO:1 enables to newly obtain and utilize a DNA that has a function (thatis, promoter activity) similar to that of the DNA comprising thenucleotide sequence represented by SEQ ID NO: 1, but which comprisesanother nucleotide sequence. Here, stringent conditions mean. conditionswherein, for example, the sodium concentration is between 10 and 300 mM,and preferably between 20 and 100 mM, and the temperature is between 25and 70° C., and preferably between 42 and 55° C.

Whether or not a variant obtained as described above or a DNA obtainedby hybridization has activity as a promoter can be confirmed bytechniques as described in the above “2. Preparation of target gene andpromoter” section.

The PDC1 promoter of the present invention can be used not only forexpressing a target gene under the control of the promoter utilizing theautoregulation mechanism, but also as a general promoter.

5. Construction of Recombinant Vector

The promoter of the present invention can be used as a general promoter,so that it can be utilized as a promoter to achieve high expression of atarget gene. The recombinant vector of the present invention can beobtained by ligating (inserting) the PDC1 promoter of the presentinvention and a target gene into an appropriate vector. Examples of “atarget gene” include a nucleic acid encoding a protein or the antisensenucleic acid thereof, a nucleic acid encoding an antisense RNA decoy anda ribozyme.

In order to produce a substance, a nucleic acid encoding a usefulprotein is preferably used as a target gene. Examples of such a usefulprotein include interferons and vaccines. In addition, a nucleic acidencoding a protein may be the nucleic acid of a gene encoding an enzymefor the production of a useful substance. An example of such a nucleicacid is the nucleic acid of a gene encoding lactate dehydrogenase forthe generation of lactic acid from pyruvic acid.

An antisense nucleic acid has a nucleotide sequence that iscomplementary to any RNA (genomic RNA and mRNA) and forms adouble-stranded chain with such RNA and thereby suppresses theexpression (transcription and translation) of gene information encodedby the RNA. As an antisense sequence, any nucleic acid substance can beused, as long as it blocks the translation or transcription of a gene.Examples of such a nucleic acid substance include a DNA, a RNA or anynucleic acid mimetics. Hence, an antisense nucleic acid(oligonucleotide) sequence is designed to be complementary to a part ofthe sequence of a gene whose expression is to be suppressed.

The length of an antisense nucleic acid sequence to be designed is notspecifically limited as long as it can inhibit the expression of a gene,and is, for example, between 10 and 50 nucleotides, and preferablybetween 15 and 25 nucleotides in length. An oligonucleotide can beeasily and chemically synthesized by a known technique.

For the purpose of the present invention, a molecular analog of anantisense oligonucleotide can also be used. The molecular analogpossesses high stability, distribution specificity and the like. Anexample of such a molecular analog is an antisense oligonucleotide towhich a chemically reactive group such as Ethylene Diamine TetraaceticAcid Iron(II) Sodium Salt Trihydrate is bound.

A nucleic acid encoding an RNA decoy indicates a gene encoding a proteinto which a transcription factor binds, or RNA having a sequence of thebinding site for a transcription factor or a sequence analogous thereto.They are introduced as “decoys” within cells, so as to suppress theaction of the transcription factor.

Ribozymes indicates an nucleic acid capable of cleaving mRNA of aspecific protein and inhibiting the translation of the specific protein.Ribozymes can be designed from a gene sequence encoding a specificprotein. For example, to design hammer-head type ribozymes, a methoddescribed in FEBS Letter, 228; 228-230 (1988) can be used. Furthermore,not only the hammer-head type ribozyme, but also those cleaving the mRNAof a specific protein, such as hairpin-type ribozymes or delta-typeribozyme, and inhibiting the translation of the specific protein can beused in the present invention.

A vector for the insertion of a target gene is not specifically limited,as long as it is a vector of a type to be integrated into a chromosome,which can integrate a target gene into the genome of a host organism asdescribed in the above “Insertion of target gene and promoter” section,or a plasmid-type vector known in the art. Examples of such a vectorinclude a plasmid DNA, a bacteriophage DNA, a retrotransposon DNA andyeast artificial chromosome DNA (YAC).

Examples of a plasmid DNA include YCp-type Escherichia coli-yeastshuttle vectors such as pRS413, pRS414, pRS415, pRS416, YCp50, pAUR112or pAUR123, YEp-type Escherichia coli-yeast shuttle vector such as pYES2or YEp13, YIp-type Escherichia coli-yeast shuttle vector such as pRS403,pRS404, pRS405, pRS406, pAUR101 or pAUR135, plasmids derived fromEscherichia coli (e.g., ColE plasmids such as pBR322, pBR325, pUC18,pUC19, pUC118, pUC119, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396or pTrc99A; p15A plasmids such as pACYC177 or pACYC184; or a pSC101plasmids such as pMW118, pMW119, pMW218 or pMW219), and plasmids derivedfrom Bacillus subtilis (e.g., pUB110 or pTP5). Examples of a phage DNAinclude λ phage (Charon4A, Charon21A, EMBL3, EMBL4, ?gt10, ?gt11 or?ZAP), fX174, M13mp18 and M13mp19. An example of retrotransposon is a Tyfactor. An example of a vector for YAC is pYACC2.

To insert the PDC1 promoter of the present invention and a target geneinto a vector, for example, a method that involves first cleaving apurified DNA with an appropriate restriction enzyme, and then insertingthe product into a restriction site or a multi-cloning site of anappropriate vector DNA, so as to ligate the product to the vector may beused.

The PDC1 promoter of the present invention should be incorporated into avector, so that it operably expresses a target gene to exert thefunction of a target gene. “Operably express” means that a target geneand the PDC1 promoter are ligated to each other, and then they areincorporated into a vector, so that the target gene is expressed underthe control of the PDC1 promoter in a host organism into which thetarget gene is inserted. Hence, to the vector of the present invention,a cis element such as an enhancer, splicing signal, poly A additionsignal, a selection marker, ribosome binding sequence (SD sequence) orthe like can be ligated, if necessary, in addition to the PDC1 promoter,a target gene and a terminator. Furthermore, examples of a selectionmarker include the dihydrofolate reductase gene, the ampicillinresistance gene and the neomycin resistance gene.

6. Transformation with Recombinant Vector

The transformant of the present invention can be obtained by introducingthe recombinant vector of the present invention into a host so that atarget gene can be expressed under the control of the PDC1 promoter. Ahost herein is not specifically limited, as long as it can express atarget gene under the control of the PDC1 promoter of the presentinvention. Examples of such hosts include bacteria belonging to thegenus Escherichia such as Escherichia coli, the genus Bacillus such asBacillus subtilis, and the genus Pseudomonas such as Pseudomonas putida.Furthermore, examples of hosts include yeast such as Saccharomycescerevisiae and Schizosaccharomyces pombe, and animal cells such as COScells and Chinese hamster ovary cell (CHO cells). Alternatively, insectcells such as Sf9 and Sf21 can also be used.

When bacteria such as Escherichia coli are used as hosts, it ispreferred that the recombinant vector of the present invention beautonomously replicable in the bacteria, and be, at the same time,composed of the promoter of the present invention, a ribosome bindingsequence, a target gene and a transcription termination sequence. Inaddition, a gene regulating the promoter of the present invention mayalso be contained.

Examples of Escherichia coli include Escherichia coli K12 and DH1, andan example of Bacillus is Bacillus subtilis. A method for introducing arecombinant vector into bacteria is not specifically limited, as long asit is a method for inserting a DNA into bacteria.

When yeast is used as a host, for example, Saccharomyces cerevisiae,Schizosaccharomyces poinbe, Pichia pastoris or the like can be used. Amethod for introducing a recombinant vector into yeast is notspecifically limited, as long as it is a method for introducing a DNAinto yeast.

When animal cells are used as hosts, simian COS-7 cells and Vero cells,CHO cells, mouse L cells, rat GH3, human FL cells or the like may beused. Examples of a method for introducing a recombinant vector intoanimal cells include an electroporation method, a calcium phosphatemethod and a lipofection method.

When an insect cell is used as a host, Sf9 cells, Sf21 cells or the likeare used. As a method for introducing a recombinant vector into aninsect cell, for example, a calcium phosphate method, a lipofectionmethod, an electroporation method or the like may be used.

When a plant is used as a host, examples of a plant include, but are notlimited to, tomato and tobacco. As a method for introducing arecombinant vector into a plant cell, for example, an agrobacteriummethod, a particle gun method, a PEG method, an electroporation methodor the like may be used.

When an insect, an animal (excluding a human) or a plant individual isused as a host, a recombinant vector can be introduced into the hostaccording to a technique known in the art for generating a transgenicanimal or plant.

Host organisms into which a recombinant vector has been introduced asdescribed above are subjected to selection for strains (clones) in whicha target gene has been introduced under the control of theabove-selected promoter. Specifically, transformant are selected usingthe above selection marker as an indicator. The thus obtainedtransformants can highly and stably express a target gene under thecontrol of the PDC1 promoter, so that the transformant can be utilizedfor producing a protein encoded by the target gene as described below,or for other purposes, such as the functional analysis of the targetgene.

7. Production of Gene Expression Product or Substance Produced byExpression Product

Next, a method for producing a gene expression product or a substanceproduced by the expression product is described. In the presentinvention, a gene expression product or a substance produced by theexpression product can be obtained by culturing the transformantobtained as described above, and collecting a gene expression product ora substance produced by the expression product from the obtainedculture. “Culture” means any of culture cells or cultured organisms, ordisrupted cells or disrupted organisms, in addition to culturesupernatant. The method of culturing the transformant of the presentinvention is performed according to a normal method applied forculturing a host.

As a medium for culturing transformants obtained using a microorganismsuch as yeast as a host, either a natural medium or a synthetic mediumcan be used, as long as the medium contains a carbon source, a nitrogensource, inorganic salts and the like that microorganisms can utilize,and enables effective culture of the transformants. As a carbon source,carbohydrate such as glucose, fructose, sucrose or starch, an organicacid such as acetic acid or propionic acid, or alcohols such as ethanolor propanol may be used. As a nitrogen source, ammonium salts ofinorganic acid or organic acid such as ammonia, ammonium chloride,ammonium sulfate, ammonium acetate or ammonium phosphate, or othernitrogen-containing compounds, as well as peptone, meat extract, cornsteep liquor or the like may be used. As an inorganic substance,potassium primary phosphate, potassium secondary phosphate, magnesiumphosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulphate, copper sulfate, calcium carbonate and the like areused.

Culture is normally performed by shake culture, culture with aerationand agitation or the like under aerobic conditions at 30° C. for 6 to 24hours. During culture, pH is maintained between 4.0 and 6.0. pH isadjusted using inorganic or organic acid, alkali solution or the like.During culture, if necessary, antibiotics such as ampicillin ortetracycline may be added to the medium.

As a medium for culturing the transformant obtained using an animal cellas a host, for example, a generally used RPMI1640 medium, DMEM medium,or any one of these media supplemented with fetal calf serum or the likeis used. Culture is normally performed in the presence of 5% CO₂ at 37°C. for 1 to 30 days. During culture, if necessary, antibiotics such askanamycin or penicillin may be added to the medium.

After the completion of culture, a gene product or a substance producedby the expression product can be collected from the culture by normalprotein purification techniques and the like. For example, when producedwithin transformed cells, the cells are disrupted by standard methodssuch as disruption by ultrasonication, trituration or disruption bypress, so as to extract a gene product or a substance produced by theexpression product. If necessary, a protease inhibitor is added.Furthermore, when the product or the substance is produced in theculture supernatant, the culture solution itself can be used.Subsequently, the solution is subjected to filtration, centrifugation orthe like to remove solid mass, and then nucleic acids are removed byprotamine suspension or the like if necessary.

Next, ammonium sulfate, alcohol, acetone or the like is added forfractionation. The precipitate is collected, and then a crude proteinsolution is obtained. The protein solution is subjected to variouschromatographies, electrophoresis or the like, thereby obtaining apurified enzyme sample. A purified gene product of interest or asubstance produced by the expression product can be obtained by, forexample, appropriately selecting from or combining gel filtration usingSephadex, Ultrogel, Biogel or the like, ion-exchange chromatography,electrophoresis using polyacrylamide gel and the like, and fractionationmethods using such as affinity chromatography or reversed-phasechromatography. However, the above culture methods and purificationmethods are examples, and the methods that can be used herein are notlimited thereto.

In addition, for example, the amino acid sequence of a purified geneproduct or a substance produced by an expression product can beconfirmed by a known method of amino acid analysis, such as an automaticmethod for determining amino acid sequences according to the Edmandegradation method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C show the construction of a chromosome-integrating typevector, pBTRP-PDC1-LDH.

FIGS. 2A to 2B show the construction of a chromosome-integrating typevector, pBTRP-PDC1-LDH.

FIG. 3 shows the genome structure of a strain that is obtained when theyeast Saccharomyces cerevisiae is transformed with vectorpBTRP-PDC1-LDH.

FIG. 4 shows the construction of chromosome-integrating type vectors,pAUR-LacZ-T123PDC1 (A), pAUR-LacZ-OC2PDC1 (B) and pAUR-LacZ-YPHPDC1 (C).

FIGS. 5A and 5B show comparison of the gene sequences of a PDC1 promoter(983 bp) isolated from a pBTRP-PDC1-LDH-introduced strain, a PDC1promoter (968 bp) isolated from IFO2260 strain, and a PDC1 promoter (968bp) isolated from YPH strain.

FIG. 6 shows β-galactosidase activity before subculture in transformantswherein the PDC1 promoter (983 bp) isolated from apBTRP-PDC1-LDH-introduced strain, the PDC1 promoter (968 bp) isolatedfrom a IFO2260 strain, and the PDC1 promoter (968 bp) isolated from aYPH strain have been inserted.

FIG. 7 shows β-galactosidase activity after subculture in transformantswherein the PDC1 promoter (983 bp) isolated from apBTRP-PDC1-LDH-introduced strain, the PDC1 promoter (968 bp) isolatedfrom a IFO2260 strain, and the PDC1 promoter (968 bp) isolated from aYPH strain have been inserted.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be further specifically described below byexamples. However, the scope of the present invention is not limited bythese examples.

EXAMPLES Example 1 Isolation of PDC1P Fragment for the Construction ofpBTRP-PDC1-LDH

In this example, the promoter region (PDC1P) of the pyruvatedecarboxylase 1 gene was determined and isolated. The PDC1P fragment wasisolated by the PCR amplification method using the genomic DNA of theSaccharomyces cerevisiae YPH strain (Stratagene) as a template.

The genomic DNA of the Saccharomyces cerevisiae YPH strain was preparedusing a Fast DNA Kit (Bio 101), which was a genome preparation kit,according to the attached protocol. The DNA concentration was measuredusing an Ultro spec 3000 spectral photometer (Amersham PharmaciaBiotech).

In a PCR reaction, Pyrobest DNA polymerase (TAKARA BIO) thought to havehigh accuracy to produce amplification fragments was used as an enzymefor amplification. The genomic DNA (50 ng/sample) of the Saccharomycescerevisiae YPH strain that had been prepared by the above technique,primer DNA (50 pmol/sample) and Pyrobest DNA polymerase (0.2units/sample) were prepared to result in a reaction of 50 μl in total.The reaction solution was subjected to a PCR amplification system (GeneAmp PCR system 9700, PE Applied Biosystems) to perform DNAamplification. Reaction conditions for the PCR amplification systemconsisted of 96° C. for 2 minutes followed by 25 cycles of 96° C. for 30seconds, 55° C. for 30 seconds and 72° C. for 90 seconds, and then 4° C.The amplification fragment of the PDC1 primer was subjected to 1% TBEagarose gel electrophoresis so as to confirm the gene amplificationfragment. In addition, the primer DNAs used for this reaction weresynthetic DNAs (Sawady Technology), and the DNA sequences of the primersare as follows.

Restriction enzyme BamHI site was added to the end of PDC1P-LDH-U (31mer and Tm of 58.3° C.) (SEQ ID NO: 2) ATA TAT GGA TCC GCG TTT ATT TACCTA TCT C

Restriction enzyme EcoRI site was added to the end of PDC1P-LDH-D (31mer and Tm or 54.4° C.) (SEQ ID NO: 3) ATA TAT GAA TTC TTT GAT TGA TTTGAC TGT G

Example 2 Construction of Recombinant Vector Containing Promoter andTarget Gene

In this example, a recombinant vector was constructed using the lactatedehydrogenase gene (LDH gene) isolated from Bifidobacterium longum as atarget gene under the control of the pyruvate decarboxylase 1 gene(PDC1) promoter sequence isolated from Saccharomyces cerevisiae.

A chromosome-integrating type vector, which had been newly constructedfor this example, was designated pBTRP-PDC1-LDH. An example of theconstruction of this vector will be described in detail below. Inaddition, an outline of this example is shown in FIGS. 1 and 2. However,the procedures for vector construction are not limited thereto.

Upon the construction of the vector, required gene fragments (a 971 bppromoter fragment (PDC1P) of the PDC1 gene and a 518 bp fragment (PDC1D)of the downstream region of the PDC1 gene) were isolated by the PCRamplification method using the genomic DNA of Saccharomyces cerevisiaeYPH strain as a template, as described above. Procedures for PCRamplification were as described above. For amplification of the fragmentof the downstream region of the PDC1 gene, the following primers wereused.

Restriction enzyme XhoI site was added to the end of PDC1D-LDH-U (34 merand Tm of 55.3° C.) (SEQ ID NO: 4) ATA TAT CTC GAG GCC AGC TAA CTT CTTGGT CGA C

Restriction enzyme ApaI site was added to the end of PDC1D-LDH-D (31 merand Tm of 54.4° C.) (SEQ ID NO: 5) ATA TAT GAA TTC TTT GAT TGA TTT GACTGT G

Each of the gene amplification fragments of PDC1P and PDC1D obtained inthe above reaction was respectively purified by ethanol precipitationtreatment. Then, the PDC1P amplification fragment and the PDC1Damplification fragment were treated by restriction enzyme reaction usingrestriction enzymes BamHI/EcoRI and restriction enzymes XhoI/ApaI,respectively. In addition, the enzymes used below were all produced byTAKARA BIO. Furthermore, detailed manuals for a series of proceduresincluding ethanol precipitation treatment and treatment with restrictionenzymes were used according to Molecular Cloning: A Laboratory Manualsecond edition (Maniatis et al., Cold Spring Harbor Laboratory press.1989).

A series of reaction procedures upon the construction of the vector wasperformed according to a general DNA subcloning method. Specifically, tothe pBluescriptII SK+ vector (TOYOBO) that had been treated withrestriction enzymes BamHI/EcoRI (TAKARA BIO) and an alkaline phosphatase(BAP, TAKARA BIO), which was a dephosphorylase, the PDC1P fragment thathad been amplified by the above PCR method and then treated withrestriction enzymes was ligated by a T4 DNA Ligase reaction (FIG. 1A).The T4 DNA Ligase reaction was performed using the LigaFast Rapid DNALigation System (Promega) according to the attached protocols.

Next, the solution that had been subjected to the ligation reaction wasthen used for the transformation of competent cells. The competent cellsused herein were Escherichia coli JM109 strain (TOYOBO), and thetransformation was performed according to the attached protocols. Theobtained culture solution was inoculated on an LB plate containing 100μg/ml antibiotics (ampicillin), followed by overnight culture. Thecolonies that had grown were confirmed by the colony PCR method using aprimer DNA of the insert fragment, and a plasmid DNA solution preparedby Miniprep was confirmed by treatment with restriction enzymes, therebyisolating the pBPDC1P vector, which was the target vector (FIG. 1B).

Subsequently, the LDH gene fragment, which had been obtained by treatingthe pYLD1 vector constructed by TOYOTA JIDOSHA KABUSHIKI KAISHA withrestriction enzymes EcoRI/AatII and T4 DNA polymerase, theterminus-modifying enzyme, was subcloned by procedures similar to thosedescribed above into the pBPDC1P vector, which had been similarlytreated with restriction enzyme EcoRI and T4 DNA polymerase, theterminus-modifying enzyme, thereby preparing the pBPDC1P-LDH I vector(FIG. 1C). In addition, the above pYLD1 vector was introduced intoEscherichia coli (name: “E. coli pYLD1”) and internationally depositedunder the Budapest Treaty and under the accession number of FERM BP-7423with the International Patent Organism Depositary at the NationalInstitute of Advanced Industrial Science and Technology (1-1-1, Higashi,Tsukuba, Ibaraki, Japan); (original deposition date: Oct. 26, 1999).Next, the vector was treated with XhoI/ApaI, and then an amplified PDC1Dfragment was ligated thereto, thereby preparing the pBPDC1P-LDH IIvector (FIG. 2A). Finally, the TRP1 marker fragment, which had beenobtained by treating the pRS404 vector (Stratagene) with AatII/SspI andT4 DNA polymerase, was ligated to the pBPDC1P-LDH II vector, which hadbeen treated with EcoRV, thereby constructing a final construct, thepBTRP-PDC1-LDH vector of a type to be introduced into a chromosome (FIG.2B).

To confirm the thus constructed chromosome-integrating typepBTRP-PDC1-LDH vector, the nucleotide sequence was determined. An ABIPRISM 310 Genetic Analyzer (PE Applied Biosystems) was used as anucleotide sequence analyzer, and determination was performed accordingto the manuals attached to this analyzer so as to discover detailsconcerning a method of preparing samples, a method of using instrumentsand the like. A vector DNA to be used as a sample was prepared by analkali extraction method. The DNA was column-purified using a GFX DNAPurification kit (Amersham Pharmacia Biotech), DNA concentration wasmeasured with an Ultro spec 3000 spectrophotometer (Amersham PharmaciaBiotech), and was then used.

Example 3 Introduction of Recombinant Vector to Host

A tryptophan dependent strain of yeast, the IFO2260 strain (the strainregistered at the Institute for Fermentation, Osaka), which was a host,was cultured in 10 ml of YPD medium at 30° C. to a logarithmic growthphase. After harvest and washing with TE buffer, 0.5 ml of TE buffer and0.5 ml of 0.2 M lithium acetate were added, and then shake culture wasperformed at 30° C. for 1 hour. Subsequently, pBTRP-PDC1-LDH, which hadbeen treated with restriction enzymes ApaI and SpeI, was added.

The suspension of the plasmid was shake-cultured at 30° C. for 30minutes, 150 ml of 70% polyethylene glycol 4000 was added, and then thesolution was agitated well. After 1 hour of shake culture at 30° C.,heat shock was given at 42° C. for 5 minutes. The cells were washed andthen suspended in 200 ml of water. The suspension was spread on aselection medium.

After the resulting colonies were isolated with the selection medium toobtain colonies, strains wherein LDH had been inserted downstream of thePDC1 promoter were obtained by PCR. Furthermore, spore formation wasperformed in media for spore formation, diploid formation was performedusing the homothallic property, and then a strain wherein the abovevector had been introduced into both chromosomes of the diploid wasobtained.

The fact that the yeast Saccharomyces cerevisiae had been transformedwith pBTRP-PDC1-LDH shown in FIG. 2 and the gene had been inserted intothe genome was confirmed by PCR. The structure of the above vector onthe genome is shown in FIG. 3.

Example 4 Production of Substance by Expression Product

The obtained transformant was inoculated at a cell concentration of 1%in YPD liquid medium (glucose 10%), and then static culture wasperformed at 30° C. for 2 days. Comparison were conducted for theamounts of lactic acid produced by (1) a strain to which no vector hadbeen introduced, (2) a strain to which LDH had been inserted with theYEP vector (a system to which LDH had been inserted under the control ofa conventional GAP promoter), and (3) a strain into which LDH had beeninserted with pBTRP-PDC1-LDH (a system into which LDH had been insertedunder the control of the PDC1 promoter). The results are shown inTable 1. TABLE 1 Comparison of the amounts of lactic acid produced as aresult of different methods of introducing LDH and subculture BeforeMethod of introducing LDH subculture After subculture (1) Parent strain(LDH was absent)   0% — (2) Introduction of LDH with YEP vector 0.4%  0% (GAP promoter) (3) Introduction of LDH with 1.0% 1.0%pBTRP-PDC1-LDH (PDC1 promoter)

While the strain (1) to which no vector had been introduced produced nolactic acid, the strains (2) and (3) into which LDH had been insertedproduced lactic acid. Furthermore, the strain (3) to which LDH had beeninserted with the chromosome-integrating type vector under the controlof the PDC1 promoter produced lactic acid at a level 2.5 times greaterthan that produced by the strain (2) into which LDH had been insertedwith the YEP vector.

Moreover, for the purpose of confirming the stability of the traitintroduced by the method, subculture was performed 3 times on YPDplates, and then gene transfer and the amount of lactic acid producedwere examined by PCR. While the system (2) into which LDH had beeninserted with the YEP vector stopped to produce lactic acid, the strain(3) which had been caused to express LDH maintained the production oflactic acid in the same amount as that produced before subculture.

Moreover, it was confirmed by PCR that there was no change in the.structure on the genome. Accordingly, the system (3) wherein LDH isexpressed by pBTRP-PDC1-LDH can be said to be present stably and enablethe high expression of the gene.

Based on the above results, it was shown that LDH is stably and highlyexpressed in the case of using the chromosome-integrating type, whereinLDH had been operably linked LDH to the PDC1 promoter of the presentinvention.

Example 5 Isolation of PDC1 Promoter Sequence Containing VariantSequence

In this example and the following examples, 3 types of PDC1 promotersequences having sequences differing in several nucleotides wereisolated. Then, chromosome-integrating type vectors designed to ligate aLacZ gene following the promoter were prepared. Transformed yeast wasconstructed by inserting the promoter and 1 copy of the gene into thesame position in the chromosome using these vectors. β-galactosidaseactivity of each transformed yeast was measured, and 3 types of promoteractivities were compared.

In this example, the PDC1 promoter sequence was isolated by the PCRamplification method using as a template the genomic DNAs of theSaccharomyces cerevisiae pBTRP-PDC1-LDH-introduced strain (the strainprepared in Example 3), the IFO2260 strain (the strain registered at theInstitute for Fermentaiton, Osaka) and the YPH strain (Stratagene).

The preparation method and the PCR amplification method for the genomicDNAs of each Saccharomyces cerevisiae strain (pBTRP-PDC1-LDH-introducedstrain, IFO2260 strain and YPH strain) were performed by techniquessimilar to those employed in Examples 1 and 2.

In addition, the nucleotide sequences of the primer DNAs used forreaction are as follows.

Amplification of the PDC1 Promoter Isolated from thepBTRP-PDC1-LDH-introduced Strain

Restriction Enzyme SalI site was added to the end of PDC1 PrFrag-U2 (32mer and Tm of 64.4° C.) (SEQ ID NO: 6) AAA TTT GTC GAC AAG GGT AGC CTCCCC ATA AC

Restriction Enzyme SalI site was added to the end of PDC1 PrFrag-D2 (31mer and Tm of 61.1° C.) (SEQ ID NO: 7) ATA TAT GTC GAC GAG AAT TGG GGGATC TTT GAmplification of IFO2260 Strain-Derived and YPH Strain-Derived PDC1Promoters

Restriction enzyme SalI site was added to the end of PDC1 PrFrag-U2 (32mer and Tm of 64.4° C.) (SEQ ID NO: 6) AAA TTT GTC GAC AAG GGT AGC CTCCCC ATA AC

Restriction Enzyme SalI Site was Added to the End of PDC1 PrFrag-D (43mer and Tm of 62.5° C.) (SEQ ID NO: 8) TTT AAA GTC GAC TTT GAT TGA TTTGAC TGT GTT ATT TTG CGT G

Example 6 Construction of Vector Containing Variant Promoter Sequencefor Analyzing β-galactosidase

In this example, under the control of the 3 types of isolated PDC1promoter sequences, vectors were constructed wherein reporter genes hadbeen ligated. As a reporter gene, β-galactosidase gene (LacZ gene) wasused.

Vectors of a type to be introduced into a chromosome that had been newlyconstructed for this example were designated pAUR-LacZ-T123PDC1,pAUR-LacZ-OC2PDC1 and pAUR-LacZ-YPHPDC1. An example of the constructionof a vector will be described in detail below. In addition, an outlineof this example is shown in FIG. 4. However, the procedures for theconstruction of the vectors are not limited to this outline.

A series of reaction procedures for the construction of the vectors wasperformed according to a general DNA subcloning method.pSV-β-Galactosidase Control Vector (Promega) was excised withrestriction enzymes so as to obtain a LacZ fragment. Then, the fragmentwas blunt-ended, so that the pAUR-LacZ vector was prepared. The thusconstructed pAUR-LacZ vector was treated with SalI (TAKARA BIO) and anAlkaline Phosphatase (BAP, TAKARA BIO), which was a dephosphorylase.Next, 3 types of promoter sequences obtained in Example 5, that is, thePDC1 promoter (983 bp) isolated from the pBTRP-PDC1-LDH-introducedstrain, the PDC1 promoter (968 bp) isolated from the IFO2260 strain, andthe PDC1 promoter (968 bp) isolated from the YPH strain, were eachtreated with restriction enzyme SalI (TAKARA BIO), and then ligated tothe pAUR-LacZ vector by a T4 DNA Ligase reaction. T4 DNA Ligase reactionwas performed using a LigaFast Rapid DNA Ligation System (Promega)according to the attached protocols.

Competent cells were transformed using the thus obtained Ligationreaction solution, and then target construction vectors were obtained bythe colony PCR method. The above series of procedures were performed bytechniques similar to those employed in Example 2.

Nucleotide sequence analysis was performed for the constructed vectors,and then the gene sequences of the PDCI promoter (983 bp) isolated fromthe pBTRP-PDC1-LDH-introduced strain, the PDC1 promoter (968 bp)isolated from the IFO2260 strain, and the PDC1 promoter (968 bp)isolated from the YPH strain were compared. The results of thecomparison of the sequences are shown in FIGS. 5A and B. In addition,nucleotide sequence analysis was performed by procedures similar to thetechniques employed in Example 2.

The PDC1promoter (983 bp) isolated from the pBTRP-PDC1-LDH-introducedstrain differed from the PDC1 promoter sequence (971 bp) comprising thenucleotide sequence represented by SEQ ID NO: 1 by 12 nucleotides.Specifically, the PDC1 promoter (983 bp) isolated from thepBTRP-PDC1-LDH-introduced strain was composed of a sequence whereinrestriction enzyme Sal I site (GTCGAC) was added to both ends of thepromoter sequence of SEQ ID NO: 1.

Furthermore, the IFO2260 strain-derived PDC1 promoter (968 bp) differedfrom the PDC1 promoter sequence comprising the nucleotide sequencerepresented by SEQ ID NO: 1 by 30 nucleotides, wherein specifically theguanine (G) at position 861 of the promoter sequence of SEQ ID NO: 1 wassubstituted with cytosine (C), the cytosine (C) at position 894 wassubstituted with thymine (T), the adenine at position 925 wassubstituted with thymine (T), and a sequence (GATCCCCCAATTCTC) of 15nucleotides was added following the nucleotide at position 972.Furthermore, the IFO2260 strain-derived PDC1 promoter sequence wascomposed of a sequence wherein restriction enzyme SalI site (GTCGAC) wasadded to both ends of the promoter sequence of SEQ ID NO: 1.

The YPH strain-derived PDC1 promoter (968 bp) differed from the PDC1promoter sequence comprising the nucleotide sequence represented by SEQID NO: 1 by 37 nucleotides, wherein, specifically, the cytosine (C) atposition 179 of the promoter sequence of SEQ ID NO: 1 was substitutedwith thymine (T), the adenine (A) at position 214 was substituted withguanine (G), the guanine (G) at position 216 was substituted withadenine (A), the thymine (T) at position 271 was substituted withcytosine (C), the guanine (G) at position 344 was substituted withadenine (A), the adenine (A) at position 490 was substituted withguanine (G), the cytosine (C) at position 533 was substituted withthymine (T), the thymine (T) at position 566 was substituted withcytosine (C), the guanine (G) at position 660 was substituted withcytosine (C), the adenine (A) at position 925 was substituted withthymine (T), and a sequence (GATCCCCCAATTCTC) of 15 nucleotides wasadded following the nucleotide at position 972. Furthermore, the YPHstrain-derived PDC1 promoter sequence was composed of a sequence whereinrestriction enzyme SalI site (GTCGAC) was added to both ends of thepromoter sequence of SEQ ID NO: 1.

Example 7 Introduction of Recombinant Vector into Host

A tryptophan dependent strain of yeast, the IFO2260 strain (the strainregistered at the Institute for Fermentation, Osaka), as a host wascultured in 10 ml of YPD medium at 30° C. to a logarithmic growth phase.After harvest and washing with TE buffer, 0.5 ml of TE buffer and 0.5 mlof 0.2 M lithium acetate were added, and then shake culture wasperformed at 30° C. for 1 hour. Subsequently, pAUR-LacZ-T123PDC1P,pAUR-LacZ-YPHPDC1P and pAUR-LacZ-OC2PDC1P, which had been treated withrestriction enzyme Bst1107 I (TAKARA BIO), were added.

The suspension of the plasmid was shake-cultured at 30° C. for 30minutes, 150 μl of 70% polyethylene glycol 4000 was added, and then thesolution was agitated well. The solution was subjected to 1 hour ofshake culture at 30° C., and then heat shock was given at 42° C. for 5minutes. The cells were cultured in 1 ml of YPD medium at 30° C. for 12hours. The culture solution was washed, and then suspended in 200 μl ofsterilized water. The suspension was then spread onto aureobasidin Aselection medium. The concentration of aureobasidin A added to themedium was 0.4 μg/ml.

The obtained colonies were isolated using the aureobasidin A selectionmedium, and then the PCR method was performed for the resultantcolonies, thereby obtaining a target strain.

Example 8 Measurement of β-galactosidase Activity in Gene RecombinantStrain

β-galactosidase activity was measured for the above transformant andnon-transformant. Each strain was cultured in 2 ml of YPD liquid medium(glucose 2%) at 30° C. for 20 hours. They were harvested and 500 μl of50 mM Tris-HCl and glass beads (425 to 600 microns Acid Washed, SIGMA)were added, and vortexed for 15 minutes at 4° C.

The supernatant of this solution was collected by centrifugation, andthen β-galactosidase activities in these supernatants were measured.Activity measurement was performed using β-Galactosidase Enzyme AssaySystem (Promega) according to the attached protocols. The value ofactivity (ABS 600 nm=1.0) was calculated, and the results are shown inFIG. 6 (before subculture) and FIG. 7 (after subculture).

Based on the above results, it was revealed that even a PDC1 promotersequence having a sequence of several tens of nucleotides added theretoor having a different sequence possesses stable promoter activity.Therefore, it can be said that the promoter of a gene wherein theautoregulation mechanism is present, or the promoter of a gene that isnot essential for growth or fermentation in a host organism can beutilized, even if it does not have a full-length sequence.

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, a gene can be stably introduced andhighly expressed in the host organism without affecting growth andfermentation of the host. Hence, an effective tools for producing asubstance and altering or analyzing the function, is provided. Moreover,according to the present invention, a promoter that activatestranscription in a host is provided. The promoter of the presentinvention enables high expression of a gene that is introduced in asmall number of copies into a host, so that it is effective to improvethe amount of a substance produced.

Sequence Listing Free Text

-   SEQ ID NO: 1: Synthetic DNA-   SEQ ID NO: 2: Synthetic DNA-   SEQ ID NO: 3: Synthetic DNA-   SEQ ID NO: 4: Synthetic DNA-   SEQ ID NO: 5: Synthetic DNA-   SEQ ID NO: 6: Synthetic DNA-   SEQ ID NO: 7: Synthetic DNA-   SEQ ID NO: 8: Synthetic DNA

1. A method of expressing a gene, which comprises inserting a targetgene into a genome under the control of the promoter of a gene whereinan autoregulation mechanism is present, or the promoter of a gene thatis not essential for growth or fermentation in a host organism.
 2. Themethod of expressing a gene of claim 1, wherein the promoter is a DNAcontaining a sequence derived from the nucleotide sequence of thepromoter of a gene in which an autoregulation mechanism is present, orthe nucleotide sequence of the promoter of a gene that is not essentialfor growth or fermentation in a host organism by deletion, substitutionor addition of 1 to 40 nucleotides, and having promoter activity.
 3. Themethod of expressing a gene of claim 1, wherein the promoter is a DNAcapable of hybridizing under stringent conditions to a DNA thatcomprises a sequence complementary to the whole or a part of thenucleotide sequence of the promoter of a gene in which an autoregulationmechanism is present, or the nucleotide sequence of the promoter of agene that is not essential for growth or fermentation in a hostorganism, and having promoter activity.
 4. The method of expressing agene of claim 1, wherein the promoter of a gene in which theautoregulation mechanism is present is the promoter of the pyruvatedecarboxylase 1 gene.
 5. The method of expressing a gene of claim 1,wherein the promoter of a gene that is not essential for growth is thepromoter of a gene encoding thioredoxin.
 6. The method of expressing agene of claim 1, wherein a host organism is any of a bacterium, a yeast,an insect, an animal or a plant.
 7. The method of expressing a gene ofclaim 6, wherein the yeast belongs to the genus Saccharomyces.
 8. Apromoter comprising any one of the following DNAs (a), (b) and (c): (a)a DNA, comprising the nucleotide sequence represented by SEQ ID NO: 1,(b) a DNA, comprising a nucleotide sequence derived from the nucleotidesequence represented by SEQ ID NO: I by deletion, substitution oraddition of I to 40 nucleotides, and having promoter activity, and (c) aDNA, capable of hybridizing under stringent conditions to a DNAcomprising a sequence complementary to the whole or a part of thenucleotide sequence represented by SEQ ID NO: 1, and having promoteractivity.
 9. A recombinant vector, containing the promoter of claim 8.10. The recombinant vector of claim 9, wherein a target gene is operablylinked to the recombinant vector.
 11. The recombinant vector of claim 9,which is a plasmid vector or a viral vector.
 12. The recombinant vectorof claim 10, wherein the target gene is any one nucleic acid selectedfrom the group consisting of a nucleic acid encoding a protein or theantisense nucleic acid thereof, a nucleic acid encoding an antisense RNAdecoy and a ribozyme.
 13. A transformant, which is obtainable bytransforming a host using the recombinant vector of any claim
 9. 14. Thetransformant of claim 13, wherein the host is a bacterium, a yeast, ananimal, an insect or a plant.
 15. The transformant of claim 14, whereinthe yeast belongs to the genus Saccharomyces.
 16. A method of producingthe. expression product of a target gene or a substance produced by theexpression product, which comprises culturing the transformant of claim13 in a medium, and collecting the expression product of a target geneor a substance produced by the expression product from the obtainedculture.
 17. A method of producing the expression product of a targetgene or a substance produced by the expression product, which comprisesculturing in a medium yeast wherein the target gene is inserted into thegenome under the control of the promoter of claim 8, and collecting theexpression product of the target gene or the substance produced by theexpression product from the obtained culture.
 18. The production methodof claim 17, wherein the target gene is inserted into the genome using arecombinant vector containing the promoter of claim
 8. 19. Theproduction method of claim 18, wherein the target gene is operablylinked to the recombinant vector.
 20. The production method of claim 18,wherein the recombinant vector is a plasmid vector or a viral vector.21. The production method of claim 17, wherein the target gene isselected from any one nucleic acid selected from the group consisting ofa nucleic acid encoding a protein or the antisense nucleic acid thereof,a nucleic acid encoding an antisense RNA decoy and a ribozyme.
 22. Theproduction method of claim 17, wherein the yeast belongs to the genusSaccharomyces.